a naphthalimide-based fluorescent probe for highly … › suppdata › cc › c4 › c4cc09488c ›...

1

Supporting Information for

A naphthalimide-based fluorescent probe for highly selective detection of histidine in aqueous solution

and its application in in-vivo imaging

Hio-Ieng Un,‡a Shuai Wu,‡b Chang-Bo Huang,a Zheng Xuc and Lin Xu*a

aShanghai Key Laboratory of Green Chemistry and Chemical Processes, Department of Chemistry, East China Normal University, 3663 N. Zhongshan Road, Shanghai 200062, China, E-mail: [email protected] Department of Changhai Hospital, The Second Military Medical University, Shanghai 200433, P. R. China.cChongqing Key Laboratory of Environmental Materials and Remediation Technology, College of Materials and Chemical Engineering, Chongqing University of Arts and Sciences, Chongqing 402160, China.

Contents:

1. General information

2. Synthesis of probe NPO and model compounds NPA and M1–3

3. The absorption and emissive properties of NPO on changing the pH

4. The absorption and emissive properties of NPC on changing the pH

5. Fluorescence emission response of NPO in the presence of various metal ions

6. Fluorescence titration of NPO with Cu2+

7. UV-Vis absorption titration spectra of NPO with Cu2+

8. Fluorescence and absorption spectra of NPC in the presence of various

amino acids

9. UV-Vis absorption titration spectra of NPC with His

10. The Job’s plots of NPO with Cu2+ and NPC with His

11.Fluorescence emission response of NPA-Cu2+ and M1-Cu2+ in the presence

of various metal ions

12.The cytotoxicity of probe NPO against HeLa cells

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2015

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1. General information

Unless otherwise mentioned, all the reagents were of analytic grade. 1H NMR and 13C NMR spectra were measured on a Bruker AM-400 spectrometer with chemical shifts reported as ppm (in CDCl3). Mass spectrometry data were obtained with a HP 5989A spectrometer. Absorption spectra were determined on a Varian Cary 100 Spectrophotometer. Fluorescence spectra were determined on a Varian Cary Eclipse.

The metal salts used were Fe(ClO4)2, Zn(ClO4)2·6H2O, Co(ClO4)2·6H2O, Ni(ClO4)2·6H2O, Ba(ClO4)2·3H2O, Pb(ClO4)2·3H2O, Cd(ClO4)2·6H2O, Cu(ClO4)2·6H2O, Mn(ClO4)2·6H2O, LiClO4·3H2O, NaClO4·H2O, AgClO4·H2O, Hg(ClO4)2·3H2O, Mg(ClO4)2·6H2O, Al(ClO4)3·9H2O.

Hela cells were obtained from Institute of Basic Medical Sciences (IBMS) of Chinese Academy of Medical Sciences (CAMS), and grown in DMEM (High glucose) medium supplemented with 10% FBS. Cells were incubated in a 5% CO2 humidified incubator at 37 oC and typically passaged with sub-cultivation ratio of 1:3 every two days.

Growing Hela Cells in the exponential phase of growth on 35-mm glass-bottom culture dishes (Φ 20mm) for 1-2 days to reach 70-90% confluency. These cells were used in fluorescence imaging experimentation. The cells were washed with DMEM for three times, and then incubated with 2 mL DMEM containing probe (10 μM) and in an atmosphere of 5% CO2 and 95% air for 30 min at 37 °C. Cells were washed twice with 1 mL PBS at room temperature, and then followed by addition of 1 mL PBS and observed under Leica TCS SP5 II confocal laser scanning microscopy using HC × PLAPO 63X oil objective (NA, 1.40), with excitation by 405 nm and 500-700 nm emission light was collected. For the Cu2+ treated samples, cells were pre-washed twice, and incubated with 10 μM probe in DMEM for 30 minutes at 37 oC. After the removal of extracellular free probes by washing for three times, the cells were then incubated with 20 μM Cu2+ for 30 minutes at 37 oC. Cells were washed twice before observation under confocal microscopy in PBS. For the histidine treated samples, cells were pre-washed twice, and incubated with 10 μM probe in DMEM for 30 minutes at 37 oC. After the removal of extracellular free probes by washing for three times, the cells were then incubated with 20 μM Cu2+ for 30 minutes at 37 oC. Cells were washed twice with PBS, and then 250 μM histidine was added and incubated for further 30 minutes. Cells were washed with PBS for three times before observation under confocal microscopy in DMEM.

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The larval stage 4 (L4) C. elegans was used. The L4 stage nematodes were washed three times with PBS. To expose the L4 stage nematodes to probe, a centrifuge tube was first filled with 1 mL of PBS buffer solution supplemented with 10 μM of probe NPO. These L4 stage worms were then incubated in the tubes at room temperature for 2.5 h. After the incubation, the exposed nematodes were washed three times again with PBS. Then the L4 stage nematodes were exposed at the Cu2+ aqueous solution, a centrifuge tube was filled with 1 mL of PBS buffer solution supplemented with 20 μM of Cu2+ which was incubated at room temperature for 3 h. For the histidine treated samples, the previously exposed worms were incubated in centrifuge tube filled with 1 mL of PBS buffer solution, containing 250 μM of histidine, at 25 °C for 3 h. The nematodes were washed three times again with PBS before being mounted onto a slide glass. Fluorescence imaging of the mounted nematodes were performed with Olympus BX51 fluorescence microscope U-FGW Ex 530−550 (Em 575 nm) channel.

The cytotoxicity of a probe NPO against HeLa cells was measured by using the standard methyl thiazolyl tetrazolium (MTT) assay. The cells were seeded into 96-well cell culture plate at 5×103/well in complete medium (DMEM containing 10% fetal bovine serum (FBS)). Cells were incubated for 24 h at 37 oC under 5% CO2 to allow attachment of the cells. After removal of the medium and washing with PBS for three times, cells were incubated with fresh medium containing various concentrations of probe for 24 h, at 37 oC under 5% CO2. Next, 80 mL fresh DMEM was added to replace the medium with different concentrations of probe, which was followed by addition of another 20 mL fresh DMEM containing 5 mg·mL-1 of MTT. After the cells were allowed to incubate with MTT for 4 h, the supernatant was removed and 150 mL DMSO was loaded into each well to dissolve the formazan under shaking for 10 min. Then the cell viability was determined by measuring the light absorbance at 570 nm with a microplate reader. Each data point was collected by averaging the values of three wells, and untreated cells were used as controls. Percentage cell viability was calculated by comparing the absorbance of the control cells to that of treated cells.

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2. Synthesis of probe NPO and model compounds NPA and M1–3

N OO

N

NH1

XCl Cl

CH3CN, K2CO3,reflux, 12 h

N OO

N

NX

Cl

2, X = N3, X = CH

N OO

Br

2-methoxyethanol,reflux, 12 h

HN NH

N OO

N

NN

Cl

H2NO


NPO

H2NO

OH


NPA

2

H2N OH


M1

N OO

N

N Cl

H2NO


M2

3

H2NO


M3

OH

The compounds NPO, NPA, M1, and M2 were prepared according to our previous

report (Chem. Asian J., 2014, 9, 3397).

Compound M3: Anhydrous potassium carbonate (138 mg, 1.0 mmol), compounds 3

(238 mg, 0.5 mmol), and 2-ethoxyethylamine (446 mg, 5.0 mmol) were dissolved in

acetonitrile (8.0 mL), and the reaction mixture was refluxed for 12 h under argon

atmosphere. The mixture was filtered, and the solvent was removed in a vacuum to

give a yellow solid. The crude product was then chromatographed on silica gel using

dichloromethane–methanol 15 : 1 (v/v) as eluant to afford 221 mg (84%) M3 as a

yellow viscous solid. 1H NMR (CDCl3, 400 MHz) δ 8.54 (d, J = 7.1 Hz, 1H), 8.47 (d,

J = 8.0 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.34 (s, 1H), 7.28

(s, 1H), 7.25 (d, J = 0.7 Hz, 1H), 7.17 (d, J = 8.1 Hz, 1H), 4.19 – 4.09 (m, 2H), 3.83 (s,

2H), 3.63 (s, 2H), 3.56 (t, J = 5.2 Hz, 2H), 3.49 (q, J = 7.0 Hz, 2H), 3.27 (s, 4H), 2.82

(t, J = 5.2 Hz, 2H), 2.75 (s, 4H), 2.29 (s, 2H), 1.68 (t, J = 7.5 Hz, 2H), 1.42 (dd, J =

15.0, 7.5 Hz, 2H), 1.18 (t, J = 7.0 Hz, 3H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (CDCl3,

100 MHz) δ: 164.49, 164.02, 155.99, 139.33, 138.02, 132.52, 131.01, 130.27, 129.84,

129.25, 128.49, 128.11, 127.41, 126.12, 125.55, 123.27, 116.65, 114.86, 69.33, 66.50,

62.98, 53.60, 53.16, 53.04, 48.62, 40.06, 30.26, 20.40, 15.19, 13.88. HR-EI-MS calcd

for C32H40N4O3 (M +): 528.3100, found: 528.3096.

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3. The absorption and emissive properties of NPO on changing the pH

360 380 400 420 440 460 480 500 520

0.00

0.04

0.08

0.12

0.16

0.20

Abso

rban

ce

Wavelength (nm)

3

3 11pH

a)

440 480 520 560 600 640 6800

100

200

300

400

500

600

700

800

3

Fluo

resc

ent I

nten

sity

(a.u

.)

Wavelength (nm)

11

pHb)

3 4 5 6 7 8 9 10 11

0

100

200

300

400

500

600

700

800

Fluo

resc

ent I

nten

sity

(527

nm

)

pH

c)

Fig. S1 The influence of pH on the absorption spectra (a) and fluorescence spectra (b) of NPO (10

µM) in water. (c) Curve of the maximum fluorescence intensity of NPO (10 μM) versus pH. (slits:

2.5, 5 nm.).

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4. The absorption and emissive properties of NPC on changing the pH

360 380 400 420 440 460 480 500 520 540

0.00

0.04

0.08

0.12

0.16

0.20

Abso

rban

ce

Wavelength (nm)

3

3 11pH

a)

440 480 520 560 600 640 6800

100

200

300

400

500

600

700

800

Fluo

resc

ent I

nten

sity

(a.u

.)

Wavelength (nm)

3

11

pH

b)

3 4 5 6 7 8 9 10 110

100

200

300

400

500

600

700

800

pH

Fluo

resc

ent I

nten

sity

(527

nm

)

c)

Fig. S2 The influence of pH on the absorption spectra (a) and fluorescence spectra (b) of NPC (10

µM) in water. (c) Curve of the maximum fluorescence intensity of NPC (10 μM) versus pH. (slits:

2.5, 5 nm.).

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5. Fluorescence emission response of NPO in the presence of various metal

ions

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Ba2+

Fe2+

Mn2+

Zn2+

Li+

Pb2+

Co2+Mg

2+

Al3+

Ag+

Ni2+

Na+Cd2+

Hg2+

F/FO

(532

nm

)

Cu2+

Fig. S3 Fluorescence emission response of NPO (10 μM) in the presence of various metal ions (all metal ions were 10 μM) in aqueous solution (10 mM HEPES, pH 7.4). (slits: 5, 5 nm).

6. Fluorescence titration of NPO with Cu2+

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

10

20

30

40

50

60

70

80

90

[Cu2+]/10-5 M

Fluo

resc

ent I

nten

sity

(532

nm

)

Fig. S4 Plot of fluorescence intensity at 532 nm of NPO versus concentration of Cu2+. (slits: 5, 5 nm).

8

7. UV-Vis absorption titration spectra of NPO with Cu2+

360 380 400 420 440 460 480 500 520 5400.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Cu2+

Abso

rban

ce

Wavelength (nm)

a)

Cu2+

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.00.000.020.040.060.080.100.120.140.160.180.20

Abso

rptio

n (m

axm

ium

)

[Cu2+]/10-5 M

b)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0350360370380390400410420430440450

Abso

rptio

n w

avel

engt

h (m

axm

ium

)

[Cu2+]/10-5 M

c)

Fig. S5 (a) UV-Vis absorption spectra of NPO (10 µM) upon addition of Cu2+ (0.5–20 μM) in aqueous solution (10 mM HEPES, pH 7.4). The curves of maximum absorption (b) and the wavelength of maximum absorbance (c) of NPO (10 μM) versus increasing concentrations of Cu2+ (0.5–20 μM). (slits: 5, 5 nm).

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8. Fluorescence and absorption spectra of NPC in the presence of various amino acids

450 500 550 600 650 7000

10

20

30

40

50

60

70

80

90

Wavelength (nm)

Fluo

resc

ent I

nten

sity

(a.u

.)

His

NPC

a)

360 380 400 420 440 460 480 500 520 5400.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Abso

rban

ce

Wavelength (nm)

His

b)

Fig. S6 Fluorescence (a) and absorption (b) spectra of NPC (the complex of 10 μM NPO and 10 μM Cu2+) in the presence of various amino acids (all amino acids 40 μM) in aqueous solution (10 mM HEPES, pH 7.4).

9. UV-Vis absorption titration spectra of NPC with His

360 380 400 420 440 460 480 500 520 5400.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Wavelength (nm)

Abso

rban

ce

His

Fig. S7 (a) UV-Vis absorption spectra of NPC (10 µM) upon addition of His (2.0–51 μM) in aqueous solution (10 mM HEPES, pH 7.4).

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10. The Job’s plots of NPO with Cu2+ and NPC with His

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.90

10

20

30

40

50

60

70

80

Fuor

esce

nce

Inte

nsity

(532

nm

)

[Cu2+]/([Cu2+]+[NPO])

a)

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.020

30

40

50

60

70

Fuor

esce

nce

Inte

nsity

(532

nm

)

[His]/([His]+[NPC])

b)

Fig. S8 (a) Job’s plot of NPO and Cu2+ ([NPO] + [Cu2+] = 10 μM) in aqueous solution (10 mM HEPES, pH 7.4). (b) Job’s plot of NPC and His ([NPC] + [His] = 40 μM) in aqueous solution (10 mM HEPES, pH 7.4).

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11. Fluorescence emission response of NPA-Cu2+ and M1-Cu2+ in the presence of various metal ions

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

(F-F

o)/F

o (5

32 n

m)

Trp

Cys

IlePro

GlnGlu

Gly

Phe

Lys

Ser

Leu

Val

ArgAl

aMe

tThr

TyrAs

p

Asn

His

a)

0

1

2

3

4

5

6

7

8

(F-F

o)/F

o (5

32 n

m)

Trp

Cys

IlePr

oGlnGl

uGl

yPh

e

LysSe

r

Leu

Val

Arg

Ala

MetTh

rTy

rAsp

Asn

His

b)

Fig. S9 Fluorescence intensity at 532 nm of (a) NPA-Cu2+ (the complex of 10 μM NPA and 10 μM Cu2+) and (b) M1-Cu2+ (the complex of 10 μM M1 and 10 μM Cu2+) in the presence of various amino acids (all amino acids 40 μM) in aqueous solution (10 mM HEPES, pH 7.4).

12

12. The cytotoxicity of probe NPO against HeLa cells

0 10 20 300

20

40

60

80

100

Cell

Viab

ility

（%

)

Concentration of NPO (M)

24h

Fig. S10 Cell viability values (%) estimated by MTT test versus incubation concentrations of NPO. Cells were incubated with 10–30 μM NPO for 24 hours.

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